Project Summary/Abstract (Overall) This application is a competitive renewal of a program project grant entitled, ?Intracellular pathogens and innate immunity.? The central problem that we address is how intracellular pathogens are recognized by the host and how the immune system integrates multiple signals to induce an appropriate response, and conversely, how pathogens avoid and/or manipulate the host response to promote their pathogenesis. In its previous iterations, this program project took advantage of newly emerging genome-wide technologies to characterize the transcriptional response of macrophages to a variety of intracellular bacterial pathogens and discovered that infection with diverse intracellular bacterial pathogens led to the activation of STING and the downstream induction of a core transcriptional response dominated by expression of type I interferon-regulated genes. To explore the host response to intracellular pathogens more deeply, each of the P01 labs has begun to investigate post-transcriptional responses to infection. In Project 1, Portnoy extends his studies on the roles of c-di-AMP and STING during Listeria monocytogenes infection and immunity. He further proposes to examine how L. monocytogenes avoids autophagy and how this impacts both pathogenesis and induction of immunity. Finally, he proposes to characterize the role of both host and bacterial protein ubiquitylation during infection. In Project 2, Cox examines how autophagosome formation is selectively activated at the Mycobacterium tuberculosis-containing phagosome. Next, based on his global ubiquitylation studies that identified IRF7 as a transcription factor that promotes the intracellular growth of M. tuberculosis, he will elucidate the mechanisms by which IRF7 promotes infection. Finally, he proposes to identify functional changes in host protein phosphorylation during M. tuberculosis infection. In Project 3, Vance proposes to disentangle the opposing effects of STING activation by using CRISPR/Cas9 technologies to generate novel STING knock-in mice that selectively eliminate specific STING-dependent responses in vivo. Next, he will determine how Legionella pneumophila targets the central metabolic regulator mTORC1, and how this affects host responses to infection. Finally, they will use comprehensive profiling technologies in Core C to identify novel post-transcriptional responses to L. pneumophila. In Core B, Barton will provide overall direction and maintain colonies of mice, breed mice, backcross mice, and use CRISPR-Cas9 to generate novel genome-edited mouse models to determine the in vivo function of post- transcriptional responses identified above. In Core C, Krogan will apply novel mass spectrometry-based and bioinformatics approaches to quantitatively profile posttranslational interactions, including ubiquitylation and phosphorylation that occur during infection. The purpose of Core A is to ensure scientific progress and promote synergy by providing scientific, organizational, and administrative leadership, which will be accomplished by extensive scientific review during monthly meetings of all the P01 lab and affiliated groups.